三个方便实用的植物表达载体构建与验证

为满足植物功能基因组学研究及转基因安全性需要,本研究根据一些国内外引进或商业化的植物表达载体及其相关元件,构建了3个适合于植物,尤其是单子叶植物转化的表达载体,即p AH006、p WMB022和p WMB025。p AH006载体包含由玉米泛素ubi启动子调控的GUS基因和bar基因的完整T-DNA区域,此区段能够被酶切回收,可用于单子叶植物农杆菌介导转化效率评价及基因枪介导线状DNA转化效果研究;p WMB022载体携带由双35S启动子调控的玉米色素基因Lc和C1,可用作基因枪介导的共转化筛选标记,直观筛选含目标基因转基因材料;p WMB025载体携带由ubi启动子调控的、商业化转基因植物中广泛利用的EPSPS基因,可用于禾谷类作物农杆菌或基因枪介导的遗传转化,载体多克隆位点可通过酶切方式更换目标基因。酶切鉴定结合农杆菌或基因枪介导的小麦幼胚愈伤组织或叶片转化验证此3个载体表明,载体构建正确,其标记基因、可视化基因和报告基因均能正常表达。这3个载体的构建对于小麦等植物转化效率提升、安全型转基因作物获得和植物功能基因组学研究等具有重要意义。

Construction and Validation of Three Convenient and Practical Constructs for Plant Transformation

To enrich tools for plant functional genomics and to develop marker-free transgenic plants, three plasmid vectors, pAH006, pWMB022 and pWMB025, ideal for cereal transformation, were constructed in this study by using some plasmids available publically. The vector pAH006 can be used to improve the Agrobacterium mediated transformation system on monocot plants and to evaluate the biolistic particle mediated transformation efficiency of linear transgene expression cassette on which both GUS and bar genes were controlled by the ubi promoters; the intact T-DNA region can be easily recovered by enzyme digestion. The vector pWMB022 carries maize pigment regulatory genes Lc and C1 under the control of double 35S promoters, which can be used to visually screen positive calli or shoots when co-bombarded with other expression vectors containing genes of interest. The vector pWMB025 carries the glyphosate-resistant gene EPSPS regulated by the ubi promoter; the vector can be used in Agrobacterium- or biolistic-mediated transformation of cereal plants to generate bio-safely transgenic materials. Genes of interest can be easily cloned into the multiple cloning site (MCS) between the ubi promoter and the nos terminator on pWMB025 by enzyme digestion. All three vectors were confirmed by enzyme digestion and then tested in Agrobacterium- or biolistic-mediated transformation by using wheat immature embryos derived calli or leaves as explants. It was shown that all three vectors were constructed successfully, and the selectable reporter/visual genes worked efficiently. Construction of these three expression vectors is important for the improvement of transformation efficiency of some recalcitrant cereals such as wheat, development of bio-safely transgenic crop varieties, and plant functional genomics.